Monoclonal antibodies specific to VEGF receptors and uses thereof

ABSTRACT

Monoclonal antibodies that specifically bind to an extracellular domain of a flt-1 receptor and neutralize activation of the receptor are provided. In vitro and in vivo methods of using these antibodies are also provided.

This application is a continuation of Ser. No. 08/967,113 filed on Nov.10, 1997, pending, which is a continuation-in-part of U.S. Pat. No.5,861,499 filed Sep. 3, 1996, which is a continuation-in-part of Ser.No. 08/476,533 filed Jun. 7, 1995, abandoned; which is a continuation ofU.S. Pat. No. 5,840,301 filed Oct. 20, 1994; which is acontinuation-in-part of Ser. No. 08/196,041 filed Feb. 10, 1994,abandoned. The entire disclosure of the aforementioned priorapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Angiogenesis is the process of developing new blood Docket Number:______ vessels that involves the proliferation, migration and tissueinfiltration of capillary endothelial cells from pre-existing bloodvessels. Angiogenesis is important in normal physiological processesincluding embryonic development, follicular growth, and wound healing aswell as in pathological conditions involving tumor growth andnon-neoplastic diseases involving abnormal neovascularization, includingneovascular glaucoma (Folkman, J. and Klagsbrun, M. Science 235:442-447(1987)).

The vascular endothelium is usually quiescent and its activation istightly regulated during angiogenesis. Several factors have beenimplicated as possible regulators of angiogenesis in vivo. These includetransforming growth factor (TGFb), acidic and basic fibroblast growthfactor (aFGF and bFGF), platelet derived growth factor (PDGF), andvascular endothelial growth factor (VEGF) (Klagsbrun, M. and D'Amore, P.(1991) Annual Rev. Physiol. 53: 217-239). VEGF, an endothelialcell-specific mitogen, is distinct among these factors in that it actsas an angiogenesis inducer by specifically promoting the proliferationof endothelial cells.

VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits withstructural similarity to PDGF. Four different monomeric isoforms of VEGFexist resulting from alternative splicing of mRNA. These include twomembrane bound forms (VEGF₂₀₆ and VEGF₁₈₉) and two soluble forms(VEGF₁₆₅, and VEGF₁₂₁). In all human tissues except placenta, VEGF₁₆₅ isthe most abundant isoform.

VEGF is expressed in embryonic tissues (Breier et al., Development(Camb.) 114:521 (1992)), macrophages, proliferating epidermalkeratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated withinflammation (Ferrara et al., Endocr. Rev. 13:18 (1992)). In situhybridization studies have demonstrated high VEGF expression in a numberof human tumor lines including glioblastoma multiforme,hemangioblastoma, central nervous system neoplasms and AIDS-associatedKaposi's sarcoma (Plate, K. et al. (1992) Nature 359: 845-848, Plate, K.et al. (1993) Cancer Res. 53: 5822-5827; Berkman, R. et al. (1993) J.Clin. Invest. 91: 153-159; Nakamura, S. et al. (1992) AIDS Weekly, 13(1)). High levels of VEGF were also observed in hypoxia inducedangiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).

The biological response of VEGF is mediated through its high affinityVEGF receptors which are selectively expressed on endothelial cellsduring embryogenesis (Millauer, B., et al. (1993) Cell 72: 835-846) andduring tumor formation. VEGF receptors typically are class IIIreceptor-type tyrosine kinases characterized by having several,typically 5 or 7, immunoglobulin-like loops in their amino-terminalextracellular receptor ligand-binding domains (Kaipainen et al., J. Exp.Med. 178:2077-2088 (1993)). The other two regions include atransmembrane region and a carboxy-terminal intracellular catalyticdomain interrupted by an insertion of hydrophilic interkinase sequencesof variable lengths, called the kinase insert domain (Terman et al.,Oncogene 6:1677-1683 (1991). VEGF receptors include FLT-1, sequenced byShibuya M. et al., Oncogene 5, 519-524 (1990); KDR, described inPCT/US92/01300, filed Feb. 20, 1992, and in Terman et al., Oncogene6:1677-1683 (1991), and FLK-1, sequenced by Matthews W. et al. Proc.Natl. Acad. Sci. USA, 88:9026-9030 (1991).

High levels of FLK-1 are expressed by endothelial cells that infiltrategliomas (Plate, K. et al., (1992) Nature 359: 845-848). FLK-1 levels arespecifically upregulated by VEGF produced by human glioblastomas (Plate,K. et al. (1993) Cancer Res. 53: 5822-5827). The finding of high levelsof FLK-1 expression in glioblastoma associated endothelial cells (GAEC)indicates that receptor activity is probably induced during tumorformation since FLK-1 transcripts are barely detectable in normal brainendothelial cells. This upregulation is confined to the vascularendothelial cells in close proximity to the tumor. Blocking VEGFactivity with neutralizing anti-VEGF monoclonal antibodies (mAbs)resulted in an inhibition of the growth of human tumor xenografts innude mice (Kim, K. et al. (1993) Nature 362: 841-844), indicating adirect role for VEGF in tumor-related angiogenesis.

Although the VEGF ligand is upregulated in tumor cells, and itsreceptors are upregulated in tumor infiltrated vascular endothelialcells, the expression of the VEGF ligand and its receptors is low innormal cells that are not associated with angiogenesis. Therefore, suchnormal cells would not be affected by blocking the interaction betweenVEGF and its receptors to inhibit angiogenesis, and therefore tumorgrowth. Blocking this VEGF-VEGF receptor interaction by using amonoclonal antibody to the VEGF receptor has not been demonstrated priorto the subject invention.

One advantage of blocking the VEGF receptor as opposed to blocking theVEGF ligand to inhibit angiogenesis, and thereby to inhibit pathologicalconditions such as tumor growth, is that fewer antibodies may be neededto achieve such inhibition. Furthermore, receptor expression levels maybe more constant than those of the environmentally induced ligandAnother advantage of blocking the VEGF receptor is that more efficientinhibition may be achieved when combined with blocking of the VEGFligand.

The object of this invention is to provide monoclonal antibodies thatneutralizes the interaction between VEGF and its receptor by binding toa VEGF receptor and thereby preventing VEGF phosphorylation of thereceptor. A further object of this invention is to provide methods toinhibit angiogenesis and thereby to inhibit tumor growth in mammalsusing such monoclonal antibodies.

SUMMARY OF THE INVENTION

The present invention provides a monoclonal antibody which specificallybinds to an extracellular domain of a VEGF receptor and neutralizesactivation of the receptor.

The invention also provides hybridoma cell lines as well as themonoclonal antibodies produced therefrom: DC101 (IgG1k) deposited asATCC Accession No. ATCC HB 11534: Mab25 (IgG1) deposited as HB12152; andMab 73 (IgG1) deposited as HB-12153.

Further, the invention provides a method of neutralizing VEGF activationof a VEGF receptor in endothelial cells comprising contacting the cellswith the monoclonal antibody of the invention.

The invention also provides a method of inhibiting angiogenesis in amammal comprising administering an effective amount of any one of theantibodies of the invention to the mammal. In addition, the inventionprovides a method of inhibiting tumor growth in a mammal comprisingadministering an effective amount of any one of the antibodies of theinvention to the mammal.

The invention also provides a pharmaceutical composition comprising anyone of the antibodies of the invention and a pharmaceutically acceptablecarrier.

DESCRIPTION OF THE FIGURES

FIG. 1: Western Blot of FLK-1/SEAPS immunoprecipitation with monoclonalantibody DC101 demonstrating that DC101 immunoprecipitates murineFLK-1:SEAPS but not SEAPS alone.

FIGS. 2A and 2B: Competitive inhibition assay indicating the effect ofanti-FLK-1 FLK-1 monoclonal antibody DC101 on VEGF₁₆₅ inducedphosphorylation of the FLK-1/fms receptor in transfected 3T3 cells. FIG.2B: Sensitivity of VEGF induced phosphorylation of the FLK-1/fmsreceptor to inhibition by monoclonal antibody DC101. C441 cells wereassayed at maximal stimulatory concentrations of VEGF₁₆₅ (40 ng/ml)combined with varying levels of the antibody.

FIGS. 3A and 3B: Titration of VEGF-induced phosphorylation of theFLK-1/fms receptor in the presence of mAb DC101. C441 cells werestimulated with the concentrations of VEGF indicated in the presence(Lanes 1 to 4) or absence (Lanes 5 to 8) of 5 ug/ml of MAb DC101.Unstimulated cells assayed in the presence of antibody (Lane 9) servesas the control. FIG. 3B: Densitometry scans of the level ofphosphorylated receptor in each lane in FIG. 3A relative to each VEGFconcentration is plotted to show the extent of Mab inhibition at excessligand concentrations. Cell lysates were prepared for detection byanti-phosphotyrosine as described in the Examples below.

FIG. 4: Inhibition of VEGF-FLK-1/fms activation by prebound mAb DC101.C441 cells were stimulated with the concentrations of VEGF indicated inthe absence (Lanes 3 and 4) and presence (Lanes 5 and 6) of DC101.Unstimulated cells (Lanes 1 and 2) serve as controls. MAb was assayedusing two sets of conditions. For P, cells were prebound with Mabfollowed by stimulation with VEGF for 15 minutes at room temperature.For C, MAb and ligand were added simultaneously and assayed as above.

FIG. 5: VEGF-induced phosphorylation of the FLK-1/fms receptor bytreatments with varying concentrations of monoclonal antibody DC101 andconditioned media from glioblastoma cells (GB CM).

FIG. 6: FACS analysis of anti-FLK-1 mAb binding to FLK-1/fms transfected3T3 Cells (C441). Transfected FLK-1/fms 3T3 cells were incubated on icefor 60 minutes with 10 ug/ml of the anti-FLK-1 MAb DC101 or the isotypematched irrelevant anti-FLK-1 MAb 23H7. Cells were washed andreincubated with 5 ug of goat anti-mouse IgG conjugated to FITC, washed,and analyzed by flow cytometry to determine antibody binding. Data showsthe level of fluorescence for DC101 to C441 cells relative to thatdetected with the irrelevant MAb 23H7.

FIG. 7: Saturation binding of mAb DC101 to the FLK-1/fms receptor on thetransfected 3T3 cell line C441. Confluent C441 cells were incubated in24 well plates with increasing concentrations of MAb DC101 (50 ng/ml to2 ug/ml) for two hours at 4° C. Cells were washed and incubated with 5ug anti-rat IgG-biotin conjugate. To detect binding, cells were washed,incubated with a 1:1000 dilution of streptavidin-HRP, washed andincubated in a colormetric detection system (TMB). Data represents theabsorbance at 540 nm versus increasing concentrations of MAb DC101. Thebinding of the secondary antibody to cells alone was subtracted fromeach determination to adjust for non-specific binding. Data representsthe average of three independent experiments.

FIG. 8: Immunoprecipitation of phosphorylated FLK-1/fms from VEGFstimulated FLK-1/fms transfected 3T3 cells. Cells were stimulated withVEGF as described in the Experimental Procedures and lysates wereimmunoprecipitated with irrelevant or relevant antibodies as follows: 1.rat anti-FLK2 IgG2a (Mab 2A13); 2. rat anti-FLK-1 IgG1 (Mab DC101); 3.rat anti-FLK2 IgG1 (Mab 23H7); 4. rabbit anti-fms polyclonal antibody.Immunoprecipitated protein was subjected to SDS PAGE followed by Westernblotting. The immunoprecipitation of VEGF activated receptor wasdetected by probing the blots with an anti-phosphotyrosine antibody.

FIG. 9: Sensitivity of VEGF-induced phosphorylation of the FLK-1/fmsreceptor to inhibition by mAb DC101. Prebound and competitive assayswere performed with 40 ng/ml of VEGF at the antibody concentrationsindicated. Cell lysates were prepared for receptor detection withanti-phophotyrosine as described in the Examples below.

FIG. 10: Effect of mAb DC101 on CSF-1 induced phosphorylation of the FMSreceptor. In (B), the fms/FLK-2 transfected 3T3 cell line, 10A2, wasstimulated with optimal stimulatory levels of CSF-1 in the absence(Lanes 3 and 4) and presence (Lanes 5 and 6) of 5 ug/ml of MAb DC101.Unstimulated cells assayed in the absence (Lane 1) or presence (Lane 2)of antibody serve as controls. Cell lysates were prepared for detectionby anti-phosphotyrosine as described in the Examples below.

FIG. 11: Specificity of mAb DC101 neutralization of the activatedFLK-1/fms receptor. C441 cells were stimulated with 20 or 40 ng/ml ofVEGF in the presence of DC101 (IgG1) or the irrelevant anti-FLK-2 ratmonoclonal antibodies 2A13 (IgG2a) or 23H7 (IgG1). Assays were performedwith each antibody in the absence of VEGF (Lanes 1 to 3) and in thepresence of VEGF under competitive (lanes 4 to 8) or prebound (lanes 9to 11) conditions. Cell lysates were prepared for detection byanti-phosphotyrosine as described in the Examples below. Blots werestripped and reprobed to detect the FLK-1/fms receptor using a rabbitpolyclonal antibody to the C-terminal region of the fms receptor.

FIG. 12: Immunoprecipitation of phosphorylated receptor bands from VEGFstimulated HUVEC cells. HUVEC cells were grown to subconfluency inendothelial growth medium (EGM) for three days without a change ofmedium. Receptor forms were immunoprecipated by MAb DC101 from lysatesof unstimulated cells (Lane 1), VEGF stimulated cells (lane 2), andcells stimulated with VEGF in the presence of 1 ug/ml heparin (Lane 3).Phosphorylation assays, immunoprecipitations, and detection of thephosphorylated receptor forms were performed as described in theExperimental Procedures.

FIG. 13: Effect of mAb DC101 on the proliferation of HUVEC cells inresponse to VEGF. Cells were grown for 48 hours as described in thelegend to FIG. 6. Cells were then subjected to the following assayconditions: no addition to medium (untreated); a change of freshendothelial growth medium (complete medium); the addition of 10 ng/ml ofVEGF in the absence or presence of 1 ug/ml heparin; and VEGF andVEGF-heparin treated cells assayed in the presence of 1 ug/ml of DC101.Cells were assayed for proliferation by colormetric detection at 550 nmusing a cell proliferation assay kit (Promega).

FIGS. 14A and 14B FIG. 14A: Reduction in tumor growth of individualanimals with DC101 (rat anti-flik-1 monoclonal antibody). FIG. 14B:Reduction in tumor growth in individual animals with the control 2A13group (rat anti-flik-2 monoclonal antibody).

FIG. 15: Athymic nude mice were injected subcutaneously with humanglioblastoma cell line GBM-18 and divided into three groups: a PBScontrol, an irrelevant rat IgG1 control 23H7, and DC101. Treatments wereadministered simultaneously with tumor xenografls and continued for fourweeks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies that bindspecifically to an extracellular domain of a VEGF receptor. Anextracellular domain of a VEGF receptor is herein defined as aligand-binding domain on extracellular region of the receptor, normallyfound at the amino-terminal end of the protein, typical of class IIItyrosine kinase receptors

Some examples of VEGF receptors include the protein tyrosine kinasereceptors referred to in the literature as FLT-1, KDR and FLK-1. Unlessotherwise stated or clearly inferred otherwise by context, thisspecification will follow the customary literature nomenclature of VEGFreceptors KDR will be referred to as the human form of a VEGF receptorhaving MW 180 kD. FLK-1 will be referred to as the murine homolog ofKDR. FLT-1 will be referred to as a form of VEGF receptor differentfrom, but related to, the KDR/FLK-1 receptor.

Other VEGF receptors include those that can be cross-link labeled withVEGF, or that can be co-immunoprecipitated with KDR (MW 180 KD). Someknown forms of these VEGF receptors have molecular weights ofapproximately 170 KD, 150 KD, 130-135 KD, 120-125 KD and 85 KD. See, forexample, Quinn et al. Proc. Nat'l. Acad. Sci 90, 7533-7537 (1993). Scheret al. J. Biol. Chem. 271, 5761-5767 (1996).

Equivalent receptors having substantially the same amino acid sequence,as defined above, occur in mammals, ie human, mouse. The binding of anantibody to one VEGF receptor does not necessarily imply binding toanother VEGF receptor, and binding to a VEGF receptor in one mammal doesnot necessarily imply binding to the equivalent receptor in anothermammal.

The VEGF receptor is usually bound to a cell, such as an endothelialcell. The VEGF receptor may also be bound to a non-endothelial cell,such as a tumor cell. Alternatively, the VEGF receptor may be free fromthe cell, preferably in soluble form.

The antibodies of the invention neutralize VEGF receptors. In thisspecification, neutralizing a receptor means inactivating the intrinsickinase activity of the receptor to transduce a signal. A reliable assayfor VEGF receptor neutralization is the inhibition of receptorphosphorylation.

The present invention is not limited by any particular mechanism of VEGFreceptor neutralization. At the time of filing this application, themechanism of VEGF receptor neutralization by antibodies is not wellunderstood, and the mechanism followed by one antibody is notnecessarilly the same as that followed by another antibody. Somepossible mechanisms include preventing binding of the VEGF ligand to theextracellular binding domain of the VEGF receptor, and preventingdimerization or oligomerization of receptors. Other mechanisms cannot,however, be ruled out.

Utility

A. Neutralizing VEGF Activation of VEGF Receptors:

Neutralization of VEGF activation of a VEGF receptor in a sample ofendothelial or non-endothelial cells, such as tumor cells, may beperformed in vitro or in vivo. Neutralizing VEGF activation of a VEGFreceptor in a sample of VEGF-receptor expressing cells comprisescontacting the cells with an antibody of the invention. In vitro, thecells are contacted with the antibody before, simultaneously with, orafter, adding VEGF to the cell sample.

In vivo, an antibody of the invention is contacted with a VEGF receptorby administration to a mammal. Methods of administration to a mammalinclude, for example, oral, intravenous, intraperitoneal, subcutaneous,or intramuscular administration.

This in vivo neutralization method is useful for inhibiting angiogenesisin a mammal. Angiogenesis inhibition is a useful therapeutic method,such as for preventing or inhibiting angiogenesis associated withpathological conditions such as tumor growth. Accordingly, theantibodies of the invention are anti-angiogenic and anti-tumorimmunotherapeutic agents.

VEGF receptors are found on some non-endothelial cells, such as tumorcells, indicating the unexpected presence of an autocrine and/orparacrine loop in these cells. The antibodies of this invention areuseful in neutralizing activity of VEGF receptors on such cells, therebyblocking the autocrine and/or paracrine loop, and inhibiting tumorgrowth.

The methods of inhibiting angiogenesis and of inhibiting pathologicalconditions such as tumor growth in a mammal comprises administering aneffective amount of any one of the invention's antibodies, including anyof the functional equivalents thereof, systemically to a mammal, ordirectly to a tumor within the mammal. The mammal is preferably human.

This method is effective for treating subjects with tumors andneoplasms, including malignant tumors and neoplasms, such as blastomas,carcinomas or sarcomas, and especially highly vascular tumors andneoplasms. Some examples of tumors that can be treated with theantibodies and fragments of the invention include epidermoid tumors,squamous tumors, such as head and neck tumors, colorectal tumors,prostate tumors, breast tumors, lung tumors, including small cell andnon-small cell Jung tumors, pancreatic tumors, thyroid tumors, ovariantumors, and liver tumors.

For example, antibodies of the invention are effective in treatingvascularized skin cancers, including squamous cell carcinoma, basal cellcarcinoma, and skin cancers that can be treated by suppressing thegrowth of malignant keratinocytes, such as human malignantkeratinocytes. Other cancers that can be treated by the antibodiesdescribed in this application include Kaposi's sarcoma, CNS neoplasms(neuroblastomas, capillary hemangioblastomas, meningiomas and cerebralmetastases), melanoma, gastrointestinal and renal carcinomas andsarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastomamultiforme, and leiomyosarcoma.

Experimental results described later demonstrate that antibodies of theinvention specifically block VEGF induced phosphorylation of a mouseextracellular FLK-1/intracellular fms chimeric receptor expressed intransfected 3T3 cells. The antibodies bad no effect on a fullystimulated chimeric extracellular fms/intracellular FLK2 receptor byCSF-1. In vivo studies also described below show that the antibodieswere able to significantly inhibit tumor growth in nude mice.

A cocktail of monoclonal antibodies of the invention provides anespecially efficient treatment for inhibiting the growth of tumor cells.The cocktail may include as few as 2, 3 or 4 antibodies, and as many as6, 8 or 10 antibodies.

The combined treatment of one or more of the antibodies of the inventionwith anti-VEGF antibodies provides a more efficient treatment forinhibiting the growth of tumor cells than the use of the antibody orantibodies alone. Anti-VEGF antibodies have been described by Kim et alin Nature 362, 841-844 (1993).

Furthermore, the combined treatment of one or more of the antibodies ofthe invention with an anti-neoplastic or chemotherapeutic drug such as,for example, doxorubicin, cisplatin or taxol provides an even moreefficient treatment for inhibiting the growth of tumor cells than theuse of the antibody by itself In one embodiment, the pharmaceuticalcomposition comprises the antibody and the anti-neoplastic orchemotherapeutic drug as separate molecules. In another embodiment, thepharmaceutical composition comprises the antibody attached, such as, forexample, by conjugation, to an chemotherapeutic drug.

Preventing or inhibiting angiogenesis is also useful to treatnon-neoplastic pathologic conditions charaqcterized by excessiveangiogenesis, such as neovascular glaucoma, proliferative retinopathyincluding proliferative diabetic retinopathy, macular degeneration,hemangiomas, angiofibromas, and psoriasis.

B. Using the Antibodies of the Invention to Isolate and Purify the VEGFReceptor

The antibodies of the present invention may be used to isolate andpurify the VEGF receptor using conventional methods such as affinitychromatography (Dean, P. D. G. et al., Affinity Chromatography: APractical Approach, IRL Press, Arlington, Va. (1985)). Other methodswell known in the art include magnetic separation with antibody-coatedmagnetic beads, “panning” with an antibody attached to a solid matrix,and flow cytometry.

The source of VEGF receptor is typically vascular cells, and especiallyvascular endothelial cells, that express the VEGF receptor. Suitablesources of vascular endothelial cells are blood vessels, such asumbilical cord blood cells, especially, human umbilical cord vascularendothelial cells (HUVEC).

The VEGF receptors may be used as starting material to produce othermaterials, such as antigens for making additional monoclonal andpolyclonal antibodies that recognize and bind to the VEGF receptor orother antigens on the surface of VEGF-expressing cells.

C. Using the Antibodies of the Invention to Isolate and Purify FLK-1Positive Tumor Cells

The antibodies of the present invention may be used to isolate andpurify FLK-1 positive tumor cells, i.e., tumor cells expressing theFLK-1 receptor, using conventional methods such as affinitychromatography (Dean, P. D. G. et al., Affinity Chromatography: APractical Approach, IRL Press, Arlington, Va. (1985)). Other methodswell known in the art include magnetic separation with antibody-coatedmagnetic beads, cytotoxic agents, such as complement, conjugated to theantibody, “panning” with an antibody attached to a solid matrix, andflow cytometry.

D. Monitoring Levels of VEGF and VEGF Receptors in Vitro or in Vivo

The antibodies of the invention may be used to monitor levels of VEGF orVEGF receptors in vitro or in vivo in biological samples using standardassays and methods known in the art. Some examples of biological samplesinclude bodily fluids, such as blood. Standard assays involve, forexample, labeling the antibodies and conducting standard immunoassays,such as radioimmunoassays, as is well know in the art.

Preparation of Antibodies

The monoclonal antibodies of the invention that specifically bind to theVEGF receptor may be produced by methods known in the art. These methodsinclude the immunological method described by Kohler and Milstein inNature 256, 495-497 (1975) and Campbell in “Monoclonal AntibodyTechnology, The Production and Characterization of Rodent and HumanHybridomas” in Burdon et al, Eds., Laboratory Techniques in Biochemistryand Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam(1985); as well as by the recombinant DNA method described by Huse et alin Science 246, 1275-1281 (1989).

The antibodies of the invention may be prepared by immunizing a mammalwith a soluble VEGF receptor. The soluble receptors may be used bythemselves as immunogens, or may be attached to a carrier protein or toother objects, such as beads, i.e. sepharose beads. After the mammal hasproduced antibodies, a mixture of antibody-producing cells, such as thesplenocytes, is isolated. Monoclonal antibodies may be produced byisolating individual antibody-producing cells from the mixture andmaking the cells immortal by, for example, fusing them with tumor cells,such as myeloma cells. The resulting hybridomas are preserved inculture, and express monoclonal antibodies, which are harvested from theculture medium.

The antibodies may also be prepared from VEGF receptors bound to thesurface of cells that express the VEGF receptor. The cell to which theVEGF receptors are bound may be a cell that naturally expresses thereceptor, such as a vascular endothelial cell. Alternatively, the cellto which the receptor is bound may be a cell into which the DNA encodingthe receptor has been transfected, such as 3T3 cells.

The antibody may be prepared in any mammal, including mice, rats,rabbits, goats and humans. The antibody may be a member of one of thefollowing immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and thesubclasses thereof, and preferably is an IgG1 antibody.

In one embodiment the antibody is a monoclonal antibody directed to anepitope of a VEGF receptor present on the surface of a cell. In anotherembodiment the monoclonal antibody is a rat IgG1 monoclonal antibody,specific for the murine VEGF receptor FLK-1, and produced by hybridomaDC101. Hybridoma cell line DC101 was deposited Jan. 26, 1994 with theAmerican Type Culture Collection, designated ATCC HB 11534. In apreferred embodiment, the monoclonal antibody is directed to an epitopeof a human FLT-1 receptor or to a human KDR receptor.

Functional Equivalents of Antibodies

The invention also includes functional equivalents of the antibodiesdescribed in this specification. Functional equivalents have bindingcharacteristics comparable to those of the antibodies, and include, forexample, chimerized, humanized and single chain antibodies as well asfragments thereof Methods of producing such functional equivalents aredisclosed in PCT Application WO 93/21319, European Patent ApplicationNo. 239,400, PCT Application WO 89/09622; European Patent Application338,745; and European Patent Application EP 332,424.

Functional equivalents include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies of the invention. “Substantiallythe same” amino acid sequence is defined herein as a sequence with atleast 70%, preferably at least about 80%, and more preferably at leastabout 90% homology to another amino acid sequence, as determined by theFASTA search method in accordance with Pearson and Lipman, Proc. Natl.Acad. Sci. USA 85, 2444-2448 (1988).

Chimerized antibodies preferably have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region from a mammal other than a human. Humanizedantibodies preferably have constant regions and variable regions otherthan the complement determining regions (CDRs) derived substantially orexclusively from the corresponding human antibody regions and CDRsderived substantially or exclusively from a mammal other than a human.

Suitable mammals other than a human include any mammal from whichmonoclonal antibodies may be made. Suitable examples of mammals otherthan a human include, for example a rabbit, rat, mouse, horse, goat, orprimate. Mice are preferred.

Functional equivalents also include single-chain antibody fragments,also known as single-chain antibodies (scFvs). Single-chain antibodyfragments are recombinant polypeptides which typically may bind withantigens or receptors; these fragments contain at least one fragment ofan antibody variable heavy-chain amino acid sequence (V_(H)) tethered toat least one fragment of an antibody variable light-chain sequence(V_(L)) with or without one or more interconnecting linkers. Such alinker may be a short, flexible peptide selected to assure that theproper three-dimensional folding of the (V_(L)) and (V_(H)) domains mayoccur once they are linked so as maintain the target moleculebinding-specificity of the whole antibody from which the single-chainantibody fragment is derived. Generally, the carboxyl terminus of the(V_(L)) or (V_(H)) sequence may be covalently linked by such a peptidelinker to the amino acid terminus of a complementary (V_(L)) and (V_(H))sequence. Single-chain antibody fragments may be generated by molecularcloning, antibody phage display library or similar techniques. Theseproteins may be produced either in eukaryotic cells or prokaryoticcells, including bacteria.

Single-chain antibody fragments contain amino acid sequences having atleast one of the variable or complentarity determining regions (CDR's)of the whole antibodies described in this specification, but are lackingsome or all of the constant domains of those antibodies. These constantdomains are not necessary for antigen binding, but constitute a majorportion of the structure of whole antibodies. Single-chain antibodyfragments may therefore overcome some of the problems associated withthe use of antibodies containing a part or all of a constant domain. Forexample, single-chain antibody fragments tend to be free of undesiredinteractions between biological molecules and the heavy-chain constantregion, or other unwanted biological activity. Additionally,single-chain antibody fragments are considerably smaller than wholeantibodies and may therefore have greater capillary permeability thanwhole antibodies, allowing single-chain antibody fragments to localizeand bind to target antigen-binding sites more efficiently. Also,antibody fragments can be produced on a relatively large scale inprokaryotic cells, thus facilitating their production. Furthermore, therelatively small size of single-chain antibody fragments makes them lesslikely to provoke an immune response in a recipient than wholeantibodies.

Functional equivalents further include fragments of antibodies that havethe same, or binding characteristics comparable to, those of the wholeantibody. Such fragments may contain one or both Fab fragments or theF(ab′)₂ fragment. Preferably the antibody fragments contain all sixcomplement determining regions of the whole antibody, although fragmentscontaining fewer than all of such regions, such as three, four or fiveCDRs, are also functional.

Further, the functional equivalents may be or may combine members of anyone of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE,and the subclasses thereof

Preparation of VEGF Receptor Immunogens

The VEGF receptor may be used as an immunogen to raise an antibody ofthe invention. The receptor peptide may be obtained from naturalsources, such as from cells that express VEGF receptors, i.e. vascularendothelial cells. Alternatively, synthetic VEGF receptor peptides maybe prepared using commercially available machines and the VEGF receptoramino acid sequence provided by, for example, Shibuya M. et al.,Oncogene 5, 519-524 (1990) for FLT-1; PCT/US92/01300 and Terman et al.,Oncogene 6:1677-1683 (1991) for KDR; and Matthews W. et al. Proc. Natl.Acad. Sci. USA, 88:9026-9030 (1991) for FLK-1.

As a further alternative, DNA encoding a VEGF receptor, such as a cDNAor a fragment thereof, may be cloned and expressed and the resultingpolypeptide recovered and used as an immunogen to raise an antibody ofthe invention. In order to prepare the VEGF receptors against which theantibodies are made, nucleic acid molecules that encode the VEGFreceptors of the invention, or portions thereof, especially theextracellular portions thereof, may be inserted into known vectors forexpression in host cells using standard recombinant DNA techniques, suchas those described below. Suitable sources of such nucleic acidmolecules include cells that express VEGF receptors, i.e. vascularendothelial cells.

Preparation of Equivalents

Equivalents of antibodies are prepared by methods known in the art. Forexample, fragments of antibodies may be prepared enzymatically fromwhole antibodies.

Preferably, equivalents of antibodies are prepared from DNA encodingsuch equivalents DNA encoding fragments of antibodies may be prepared bydeleting all but the desired portion of the DNA that encodes the fulllength antibody.

DNA encoding chimerized antibodies may be prepared by recombining DNAsubstantially or exclusively encoding human constant regions and DNAencoding variable regions derived substantially or exclusively from thesequence of the variable region of a mammal other than a human. DNAencoding humanized antibodies may be prepared by recombining DNAencoding constant regions and variable regions other than thecomplementarity determining regions (CDRs) derived substantially orexclusively from the corresponding human antibody regions and DNAencoding CDRs derived substantially or exclusively from a mammal otherthan a human.

Suitable sources of DNA molecules that encode fragments of antibodiesinclude cells, such as hybridomas, that express the full lengthantibody. The fragments may be used by themselves as antibodyequivalents, or may be recombined into equivalents, as described above.

The DNA deletions and recombinations described in this section may becarried out by known methods, such as those described in the publishedpatent applications listed above in the section entitled “FunctionalEquivalents of Antibodies” and/or other standard recombinant DNAtechniques, such as those described below.

Standard Recombinant DNA Techniques

Standard recombinant DNA techniques are described in Sambrook et al.,“Molecular Cloning,” Second Edition, Cold Spring Harbor Laboratory Press(1987) and by Ausubel et al. (Eds) “Current Protocols in MolecularBiology,” Green Publishing Associates/Wiley-Interscience, New York(1990).

Briefly, a suitable source of cells containing nucleic acid moleculesthat express the desired DNA, such as an antibody, antibody equivalentor VEGF receptor, is selected. See above.

Total RNA is prepared by standard procedures from a suitable source. Thetotal RNA is used to direct cDNA synthesis. Standard methods forisolating RNA and synthesizing cDNA are provided in standard manuals ofmolecular biology such as, for example, those described above.

The cDNA may be amplified by known methods. For example, the cDNA may beused as a template for amplification by polymerase chain reaction (PCR);see Saiki et al., Science, 239, 487 (1988) or Mullis et al., U.S. Pat.No. 4,683,195. The sequences of the oligonucleotide primers for the PCRamplification are derived from the known sequence to be amplified. Theoligonucleotides are synthesized by methods known in the art. Suitablemethods include those described by Caruthers in Science 230, 28 1-285(1985).

A mixture of upstream and downstream oligonucleotides are used in thePCR amplification. The conditions are optimized for each particularprimer pair according to standard procedures. The PCR product isanalyzed, for example, by electrophoresis for cDNA having the correctsize, corresponding to the sequence between the primers.

Alternatively, the coding region may be amplified in two or moreoverlapping fragments. The overlapping fragments are designed to includea restriction site permitting the assembly of the intact cDNA from thefragments.

In order to isolate the entire protein-coding regions for the VEGFreceptors, for example, the upstream PCR oligonucleotide primer iscomplementary to the sequence at the 5′ end, preferably encompassing theATG start codon and at least 5-10 nucleotides upstream of the startcodon. The downstream PCR oligonucleotide primer is complementary to thesequence at the 3′ end of the desired DNA sequence. The desired DNAsequence preferably encodes the entire extracellular portion of the VEGFreceptor, and optionally encodes all or part of the transmembraneregion, and/or all or part of the intracellular region, including thestop codon.

The DNA to be amplified, such as that encoding antibodies, antibodyequivalents, or VEGF receptors, may also be replicated in a wide varietyof cloning vectors in a wide variety of host cells. The host cell may beprokaryotic or eukaryotic.

The vector into which the DNA is spliced may comprise segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Some suitableprokaryotic cloning vectors include plasmids from E. coli, such ascolE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors alsoinclude derivatives of phage DNA such as M13 and other filamentoussingle-stranded DNA phages.

A preferred vector for cloning nucleic acid encoding the VEGF receptoris the Baculovirus vector.

The vector containing the DNA to be expressed is transfected into asuitable host cell. The host cell is maintained in an appropriateculture medium, and subjected to conditions under which the cells andthe vector replicate. The vector may be recovered from the cell. The DNAto be expressed may be recovered from the vector.

Expression and Isolation of Antibodies Antibody Equivalents or VEGFReceptors

The DNA to be expressed, such as that encoding antibodies, antibodyequivalents, or VEGF receptors, may be inserted into a suitableexpression vector and expressed in a suitable prokaryotic or eucaryotichost cell.

For example, the DNA inserted into a host cell may encode the entireextracellular portion of the VEGF receptor, or a soluble fragment of theextracellular portion of the VEGF receptor. The extracellular portion ofthe VEGF receptor encoded by the DNA is optionally attached at either,or both, the 5′ end or the 3′ end to additional amino acid sequences.The additional amino acid sequences may be attached to the VEGF receptorextracellular region in nature, such as the leader sequence, thetransmembrane region and/or the intracellular region of the VEGFreceptor. The additional amino acid sequences may also be sequences notattached to the VEGF receptor in nature. Preferably, such additionalamino acid sequences serve a particular purpose, such as to improveexpression levels, secretion, solubility, or immunogenicity.

Vectors for expressing proteins in bacteria, especially E. coli, areknown. Such vectors include the PATH vectors described by Dieckmann andTzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectors containDNA sequences that encode anthranilate synthetase (TrpE) followed by apolylinker at the carboxy terminus. Other expression vector systems arebased on beta-galactosidase (pEX); lambda P_(L); maltose binding protein(pMAL); and glutathione S-transferase (pGST)—see Gene 67, 31 (1988) andPeptide Research 3, 167 (1990).

Vectors useful in yeast are available. A suitable example is the 2μplasmid.

Suitable vectors for expression in mammalian cells are also known. Suchvectors include well-known derivatives of SV-40, adenovirus,retrovirus-derived DNA sequences and shuttle vectors derived fromcombination of functional mammalian vectors, such as those describedabove, and functional plasmids and phage DNA.

Further eukaryotic expression vectors are known in the art (e.g., P. J.Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S.Subramani et al, Mol. Cell. Biol. 1, 854-864 (1981); R. J. Kaufmann andP. A. Sharp, “Amplification And Expression Of Sequences Cotransfectedwith A Modular Dihydrofolate Reductase Complementary DNA Gene,” J. Mol.Biol. 159, 601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell.Biol. 159, 601-664 (1982); S. I. Scahill et al, “Expression AndCharacterization Of The Product Of A Human Immune Interferon DNA Gene InChinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA 80, 4654-4659(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA 77,4216-4220, (1980).

The expression vectors useful in the present invention contain at leastone expression control sequence that is operatively linked to the DNAsequence or fragment to be expressed. The control sequence is insertedin the vector in order to control and to regulate the expression of thecloned DNA sequence. Examples of useful expression control sequences arethe lac system, the trp system, the tac system, the trc system, majoroperator and promoter regions of phage lambda, the control region of fdcoat protein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., Pho5, the promoters of the yeast alpha-mating factors, andpromoters derived from polyoma, adenovirus, retrovirus, and simianvirus, e.g., the early and late promoters or SV40, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells and their viruses or combinations thereof

Vectors containing the control signals and DNA to be expressed, such asthat encoding antibodies, antibody equivalents, or VEGF receptors, areinserted into a host cell for expression. Some useful expression hostcells include well-known prokaryotic and eukaryotic cells. Some suitableprokaryotic hosts include, for example, E. coli, such as E coli SG-936,E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coliDHI, and E. coli MRCl, Pseudomonas, Bacillus, such as Bacillus subtilis,and Streptomyces. Suitable eukaryotic cells include yeast and otherfungi, insect, animal cells, such as COS cells and CHO cells, humancells and plant cells in tissue culture.

Following expression in a host cell maintained in a suitable medium, thepolypeptide or peptide to be expressed, such as that encodingantibodies, antibody equivalents, or VEGF receptors, may be isolatedfrom the medium, and purified by methods known in the art. If the, thepolypeptide or peptide is not secreted into the culture medium, the hostcells are lysed prior to isolation and purification.

EXAMPLES

The Examples which follow are set forth to aid in understanding theinvention but are not intended to, and should not be construed to, limitits scope in any way. The Examples do not include detailed descriptionsof conventional methods, such as those employed in the construction ofvectors and plasmids, the insertion of genes encoding polypeptides intosuch vectors and plasmids, or the introduction of plasmids into hostcells. Such methods are well known to those of ordinary skill in the artand are described in numerous publications including Sambrook, J.,Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A LaboratoryManual, 2nd edition, Cold Spring Harbor Laboratory Press.

Example I Cell Lines and Media

NIH 3T3 cells were obtained from the American Type Culture Collection(Rockville Md,). The C441 cell line was constructed by transfecting 3T3cells with the chimeric receptor mouse FLK1/human fins. 10A2 is a 3T3transfectant containing the chimeric receptor human fms/mouse FLK2, theisolation and characterization of which has been described (Dosil, M. etal., Mol. Cell. Biol. 13:6572-6585 (1993)). Cells were routinelymaintained in Dulbecco's modified Eagle's medium (DME) supplemented with10% calf serum (CS), 1 mM L-glutamine, antibiotics, and 600 ug/ml G418(Geneticin; Sigma, St Louis Mo.).

A glioblastoma cell line, GBM-18, was maintained in DME supplementedwith 5% calf serum, 1 mM L-glutamine, and antibiotics.

A stable 3T3 line secreting the soluble chimeric protein, mouseFLK1:SEAPs (secretory alkaline phosphastase), was generated andmaintained in DMEM and 10% calf serum. Conditioned media was collected.Soluble FLK-1:SEAP is isolated from the conditioned media.

Example II Isolation of Monoclonal Antibodies Example II-1 Rat AntiMouse FLK-1 Monoclonal Antibody DC101 (IgG1)

Lewis rats (Charles River Labs) were hyperimmunized with an immunecomplex consisting of the mouse FLK-1 :SEAPs soluble receptor, a rabbitanti-alkaline phosphatase polyclonal antibody and Protein-G sepharosebeads. The animals received 7 intraperitoneal injections of this complexspread over three months (at days 0, 14, 21, 28, 49, 63, 77). At varioustimes, the animals were bled from the tail vein and immune sera screenedby ELISA for high titer binding to mFLK-1:SEAPs. Five days after thefinal injection, rats were sacrificed and the spleens asepticallyremoved. Splenocytes were washed, counted, and fused at a 2:1 ratio withthe murine myeloma cell line NS1. Hybridomas were selected in HAT mediumand colonies screened by ELISA for specific binding to mFLK-1 :SEAPs butnot the SEAPs protein. A number of positive hybridomas were expanded andcloned three times by limiting dilution. One subclone, designated DC101,was further characterized.

Example II-2 Mouse Anti Mouse FLK-1 Monoclonal Antibodies Mab 25 and Mab73

Murine anti-FLK-1 monoclonal antibodies (Mabs) were produced using asimilar protocol as that employed for DC101. Briefly, mice were injectedwith a complex of FLK-1/SEAP soluble receptor bound to either ananti-SEAP-Protein/A Sepharose complex or wheat germ agglutinin Sepharosefrom conditioned medium of transfected NIH 3T3 cell. Mice werehyperimmunized at periodic intervals over a 6 month period. Immunesplenocytes were pooled and fused with the murine myeloma cell line,NSI. Hybidomas were selected in HAT medium and following incubation,colonies were screened for mouse Mab produciton. Unlike the protocolemployed for DC101, positive supernatants were initially screened forbinding to the FLK-1/fms receptor captured from C441 cell lysates onELISA plates coated witha peptide generated polyclonal antibody againstthe C-terminal region of fms. Reactive Mabs were then assayed by ELISAfor binding to intact C441 cells and to purified FLK-1/SEAP versus SEAPalone. The supernatants from hybridomas showing binding to C441 andreactivity with FLK-1/SEAP but not SEAP were expanded, grown in ascites,and purified (EZ-PREP, Pharmacia). Purified Mabs were subjected toassays on C441 cells to determine their cell surface binding by FACS andtheir ability to inhibit VEGF induced activation of FLK-1/fms inphosphorylation assays. The results of these studies led to the cloningof Mabs 25 and 73 (isotype IgG1) for further characterization based ontheir capabilities to bind specifically to FLK-1 and block receptoractivation at levels comparable to that observed for DC101.

Example III Assays Example III-1 ELISA Methods

Antibodies were screened by a solid state ELISA in which the bindingcharacteristics of the various mAbs to FLK-1 :SEAP and SEAP protein werecompared. Microtiter plates were coated with 50-100 ng/well of eitherFLK-1 :SEAP or AP in pH9.6 carbonate buffer overnight at 4° C. Plateswere blocked with phosphate buffered saline supplemented with 10% newborn calf serum (NB10) for one hour at 37° C. Hybridoma supernatants orpurified antibodies were added to the plates for two hours at 37° C.followed by goat anti-rat IgG conjugated to horse radish peroxidase(Tago) added for an additional hour at 37° C. After extensive washing,TMB (Kirkegaard and Perry, Gaithersburg Md.) plus hydrogen peroxide wasadded as the chromogen and the plates read at 450 nm in an ELISA reader.

Example III-2 Isotyping

Isotyping of the various monoclonal antibodies was done as previouslydescribed (Songsakphisarn, R. and Goldstein, N. I., Hybridoma 12:343-348, 1993) using rat isotype specific reagents (Zymed Labs, SouthSan Francisco Calif.).

Example III-3 Phosphorylation, Immunoprecipitation and Immunoblot Assays

The phosphorylation assays and Western blot analysis with C441 and 10A2cells were performed as previously described (Tessler et al., 1994) withsome modifications. Briefly, cells were grown to 90% confluency inDME-10% CS and then serum starved in DME-0.5% CS for 24 hours prior toexperimentation. HUVEC cells were grown to subconfluence in EGM basalmedia. For neutralization assays, cells were stimulated with variousconcentrations of the appropriate ligand under serum free conditions(DME -0.1% BSA) in the presence and absence of mAb DC101 for 15 minutesat room temperature. The ligands, VEGF and CSF-1, were assayed atconcentrations of 10-80 ng/ml and 20-40 ng/ml, respectively. Monoclonalantibodies were assayed at concentrations ranging from 0.5 ug/ml to 10ug/ml. To evaluate the effects of mAb DC101 on the VEGF inducedactivation of the FLK-1-fms receptor, antibody was either addedsimultaneously (competitive inhibition) or prebound to cells for 15minutes at room temperature prior to the addition of ligand. Cellsincubated in serum free medium in the absence and presence of DC101served as controls for receptor autophosphorylation in the absence ofligand and the presence of antibody, respectively. A control cell lineexpressing the fms/FLK2 chimeric receptor (10A2) was starved andstimulated with 20 and 40 ng/ml CSF-1 and assayed in the presence andabsence of 5 ug/ml DC101.

Following stimulation, monolayers were washed with ice cold PBScontaining 1 mM sodium orthovanadate. Cells were then lysed in lysisbuffer (20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 137 mM NaCl, 10%glycerol, 10 mM EDTA, 2 mM sodium orthovanadate, 100 mM NaF, 100 mMsodium pyrophosphate, 5 mM Pefabloc (Boehringer Mannheim Biochemicals,Indianapolis Ind.), 100 ug aprotinin and 100 ug/ml leupeptin) andcentrifuged at 14000×g for 10 minutes. Protein was immunoprecipitatedfrom cleared lysates of transfected cells using polyclonal antibodiesgenerated to peptides corresponding to the C-terminal region of thehuman fms receptor (Tessler et al., J. Biol. Chem. 269, 12456-12461,1994) or the murine FLK-2 interkinase domain (Small et al., Proc. Natl.Acad. Sci. USA, 91, 459-463, 1994) coupled to Protein A Sepharose beads.Where indicated, immunoprecipitalions with DC101 or irrelevant rat IgGwere performed with 10 ug of antibody coupled to Protein G beads. Thebeads were then washed once with 0.2% Triton X-100, 10 mM Tris-HClpH8.0, 150 mM NaCl, 2 mM EDTA (Buffer A), twice with Buffer A containing500 mM NaCl and twice with Tris-HCl, pH8.0. Drained beads were mixedwith 30 ul in 2× SDS loading buffer and subjected to SDS PAGE in 4-12%gradient gels (Novex, San Diego Calif.). After electrophoresis, proteinswere blotted to nitrocellulose filters for analysis. Filters wereblocked overnight in blocking buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl(TBS) containing 5% bovine serum albumin and 10% nonfat dried milk(Biorad, Calif.). To detect phosphorylated receptor, blots were probedwith a monoclonal antibody directed to phosphotyrosine (UBI, LakePlacid, N.Y.) in blocking buffer for 1 hour at room temperature. Blotswere then washed extensively with 0.5× TBS containing 0.1% Tween-20(TBS-T) and incubated with goat anti-mouse Ig conjugated to horseradishperoxidase (Amersham). Blots were washed with TBS and incubated for 1minute with a chemiluminescence reagent (ECL, Amersham).Anti-phosphotyrosine reacting with phosphorylated proteins was detectedby exposure to a high performance luminescence detection film(Hyperfilm-ECL, Amersham) for 0.5 to 10 minutes.

To detect FLK-1/fms in C441 cells receptor levels, blots were strippedaccording to manufacturer's protocols (Amersham) and reprobed with theanti-fins rabbit polyclonal antibody.

Example III-4 Flow Cytometer Binding Assays

C441 cells were grown to near confluency in 10 cm plates. Cells wereremoved with a non-enzymatic dissociation buffer (Sigma), washed in coldserum free medium and resuspended in Hanks balanced salt solutionsupplemented with 1% BSA (HBSS-BSA) at a concentration of 1 millioncells per tube. mAb DC101 or an isotype matched irrelevant antibody antiFLK-2 23H7 was added at 10 ug per tube for 60 minutes on ice. Afterwashing, 5ul of goat anti-mouse IgG conjugated to FITC (TAGO) was addedfor an additional 30 minutes on ice. Cells were washed three times,resuspended in 1 ml of HBSS-BSA, and analyzed on a Coulter Epics EliteCytometer. Non-specific binding of the fluorescent secondary antibodywas determined from samples lacking the primary antibody.

Example III-5 Binding Assays to Intact Cells

Assays with C441 cells were performed with cells grown to confluency in24 well dishes. C cells were grown to confluency in 6 well dishes.Monolayers were incubated at 4° C. for 2 hours with various amounts ofmAb DC101 in binding buffer (DMEM, 50 Mm HEPES pH 7.0, 0.5% bovine serumalbumin). Cells were then washed with cold phosphate buffered saline(PBS) and incubated with a secondary anti-rat IgG antibody conjugatedwith biotin at a final concentration of 2.5 ug/ml. After 1 hour at 4° C.cells were washed and incubated with a streptavidin-horse radishperoxidase complex for 30 minutes at 4° C. Following washing, cell-boundantibody was determined by measuring the absorbance at 540 nm obtainedwith a colormetric detection system (TMB, Kirkegaard and Perry). The OD540 nm of the secondary antibody alone served as the control fornon-specific binding.

Example III-6 Cell Proliferation Assays

Mitogenic assays were performed using the Cell Titer 96 Non RadioactiveCell Proliferation Assay Kit (Promega Corp., Madison, Wis.). In thisassay proliferation is measured color metrically as the value obtainedfrom the reduction of a tetrazolium salt by viable cells to a formazanproduct. Briefly, HUVEC cells were grown in 24 well gelatin-coatedplates in EGM basal media at 1000 cells/well. After a 48-hour incubationvarious components were added to the wells. VEGF was added at 10 ng/mlto the media in the presence and absence of 1 ug/ml of mAb DC101. Whereindicated, heparin (Sigma) was added to a final concentration of 1ug/ml. Cells were then incubated for an additional 3 days. To measurecell growth, a 20 ul aliquot of tetrazolum dye was added to each welland cells were incubated for 3 hrs at 37° C. Cells were solubilized andthe absorbance (OD570) of the formazan product was measured as aquantitation of proliferation.

Example IV In vitro Activity Assays Example IV-1 Murine Anti-FLK-1 Mabs25 and 73 Elicit a Specific Neutralization of VEGF Induced Activation ofthe FLK-1/fms Receptor

Assays were performed with immunoprecipitated FLK/fms and PDGF receptorsfrom equal concentrations of the FLK-1/fms transfected 3T3 cell line,C441 whereas the human EGFR was immunoprecipitated from the tumor cellline, KB. Cells were stimulated with RPMI-0.5% BSA containing 20 ng/mlVEGF (FLK-1/fms), DMEM-10% calf serum (PDGFR), or 10 ng/ml EGF (EGFR),in the presence and absence of 10 ug/ml of the murine anti-FLK-1 Mabs,25 and 73. Following stimulation, cells were washed with PBS-1 mM sodiumorthovanadate and lysed. FLK-1/fms and PDGFR were immunoprecipitatedfrom lysates with peptide generated polyclonal antibodies against theC-terminal region of the c-fms (IM 133) and the PDGF (UBI) receptors,respectively. EGFR was immunoprecipitated with a Mab (C225) raisedagainst the N-terminal region of the human receptor. Immunoprecipitatedlystates were subjected to SDS polyacrylamide electrophoresis followedby western blotting. Blots were probed with an anti-PTyr Mab (UBI) todetect receptor activation. Receptor neutralization of stimulated cellswas assessed relative to an irrelevant Mab and the unstimulated control.

Example IV-2 Detection of the FLK-1/fms Receptor by Western Blottingusing Mab 25 and Mab 73 as Probes

Receptor was detected by the murine anti-FLK-1 Mabs on western blots ofthe FLK-1/fms receptor immunoprecipiated by a peptide generatedpolyclonal antibody against the C-terminal region of the c-fms receptorfrom lysated prepared from equal concentrations of transfected 3T3 cellline C441. Following analysis by SDS gel electrophoresis and westernblotting, the blot was divided into four parts and each section wasprobed with 50 ug/ml of the anti-FLK-1 Mabs 25 and 73. Blots were thenstripped and reprobed with the anti-fms polyclonal antibody to verifythat the bands deted by each Mab represented the FLK-1/fms receptor.

Example IV-3 Detection of Activated KDR from VEGF Stimulated HUVEC andOVCAR-3 Cells by Immunoprecipiation with Anti-FLK-1 Mabs

Proteins were immunoprecipitated by different antibodies from a lysateof freshly isolated HUVEC. Prior to lysis, cells were stimulated with 20ng/ml VEGF for 10 minutes at room temperature in RPMI-0.5% BSA andwashed with PBS containing 1 mM sodium orthovanadate. Individualimmunoprecipitations were performed with equal volumes of lysate andthen subjected to SDS polyacrylarnide electrophoresis followed bywestern blotting. The blot was probed initially with an anti-PTyr Mab(UBI) and then sequentially stripped and reprobed with a peptidegenerated polyclonal antibody against the interkinase of FLK-1/KDR (IM142), followed by a polyclonal antibody against the C-terminal region ofFLT-1 (Santa Cruz Biotechnology, Inc). The immunoprecipitations wereperformed with an irrelevant rat Mab, 23H7, an irrelevant mouse Mab, DAB8, versus the anti-FLK-1 Mabs, DC101, 73, 25 and an anti-FLK-1/KDRpolyclonal antibody, IM 142. In some cases blots were stripped andreprobed with the anti-FLK-1 Mabs 73 and 25 to detect cross reactivebands.

A similar protocol was employed to detect KDR receptor form(s) in theovarian carcinoma cell line OVCAR-3.

Example V Activity of Antibodies Example V-1 ELISA andImmunoprecipitation with DC101

Rat IgG1 monoclonal antibody DC101 was found to be specific for themurine tyrosine kinase receptor FLK-1. ELISA data showed that theantibody bound to purified FLK-1:SEAP but not alkaline phosphatase orother receptor tyrosine kinases such as FLK-2. As seen in FIG. 1, DC101immunoprecipitates murine FLK-1:SEAPS but not SEAPS alone.

Example V-2 Inhibition of FLK-1 Receptor Phosphorylation with DC101

Experiments were then performed to determine whether DC101 couldneutralize phosphorylation of FLK1 in C441 cells by its cognate ligand,VEGF₁₆₅. In these studies, monoclonal antibody and VEGF were addedsimultaneously to monolayers for minutes at room temperature. Theseconditions were designed to determine the competitive effects(competitive inhibition) of the antibody on receptor/ligand binding. Theresults of these assays, shown in FIG. 2A, indicate that VEGF₁₆₅ inducedphosphorylation of the FLK1/fms receptor was markedly reduced when cellswere assayed in the presence of DC101. In addition, these data suggestthat the Mab competes with VEGF₁₆₅ to prevent a full activation ofreceptor by ligand. To determine the sensitivity of the VEGF-FLK1interaction to inhibition by DC101, C441 cells were assayed at maximalstimulatory concentrations of VEGF₁₆₅ (40 ng/ml) combined with varyinglevels of the antibody. The results of these Mab titrations are shown inFIG. 2B. A marked decrease in the phosphorylation of FLK1 by VEGF₁₆₅ wasobserved when DC101 was included at concentrations greater than 0.5ug/ml. These data show that relatively low concentrations of antibody(<1 ug/ml) are sufficient to inhibit receptor activation by ligand. At 5ug/ml the antibody is able to neutralize VEGF₁₆₅ stimulation of FLK1 inthe presence of excess ligand at 80 ng/ml (FIGS. 3A and 3B). As acontrol, the effect of DC101 was tested on the fully stimulated fms/FLK2receptor (10A2 cell line) using CSF-1. Under these conditions, DC101showed no effect on receptor activation.

Example V-3 Inhibition Studies with DC101

The extent and specificity of Mab inhibition was further assessed bystudies in which DC101 was preincubated with cells before the additionof ligand to allow maximal interaction of antibody with receptor. Inthese experiments, monolayers were incubated with 5 ug/ml of DC101, arat anti-FLK2 Mab (2A13) prepared by conventional techniques (ImClone,NY), and control rat IgG1 (Zymed Labs) for 15 minutes at roomtemperature prior to the addition of 40 ng/ml of VEGF₁₆₅ for anadditional 15 minutes. For comparison, assays were run in which DC101and VEGF₁₆₅ were added simultaneously (competitive inhibition). Theresults of these studies (FIG. 4) show that preincubation of theanti-FLK-1 monoclonal antibody with FLK1/fms transfected cellscompletely abrogates receptor activation by VEGF₁₆₅. Similar resultswere observed using VEGF₁₂₁ for stimulation. While phosphorylation ofFLK1 by VEGF is not affected by the addition of irrelevant isotypematched rat antibodies, the reactivity of the same blot probed with theanti-fms polyclonal antibody shows an equal level of receptor proteinper lane. These data indicate that the inhibition of phosphorylationobserved with DC101 was due to the blockage of receptor activationrather than a lack of receptor protein in the test samples.

Example V-4 Binding of DC101 to C441 Cells by FACS Analysis

The mAb was assayed by FACS analysis for binding to 3T3 cellstransfected with the FLK-1/fms receptor (C441 cells). The results, shownin FIG. 6, demonstrate that the chimeric FLK-1/fins expressed on thesurface of C441 cells is specifically recognized by mAb DC101 and not byan antibody of the same isotype raised against the related tyrosinekinase receptor, FLK-2. The efficacy of the mab-receptor interaction atthe cell surface was determined from assays in which varying levels ofmAb binding was measured on intact C441 cells. These results, shown inFIG. 7, indicate that mAb binds to the FLK-1/fms receptor with arelative apparent affinity of approximately 500 ng/ml. These resultsindicate that the mAb has a strong affinity for cell surface expressedFLK-1.

Example V-5 Reactivity of DC101 by Immunoprecipitation

The extent of DC101 reactivity with the FLK-1/fms receptor was furtherassessed by determining the capacity of the antibody toimmunoprecipitate the receptor following activation by VEGF. FIG. 8shows an immunoprecipitation by mAb DC101 of the phosphorylatedFLK-1/fms receptor from VEGF stimulated C441 cells. The results showthat the DC101 monoclonal and anti-fms polyclonal antibodies displaysimilar levels of receptor interaction while rat anti FLK-2 antibodies2H37 (IgG1) and 2A13 (IgG2a) show no reactivity. Experiments were thenperformed to determine whether mAb DC101 could neutralize the VEGFinduced phosphorylation of FLK-1/fins at maximal stimulatoryconcentrations of ligand (40 ng/ml). In these studies, monoclonalantibody was added to monolayers either simultaneously with ligand orprior to ligand stimulation and assayed for 15 minutes at roomtemperature. These conditions were studied to determine both thecompetitive effects (competitive inhibition) of the antibody onreceptor/ligand binding as well as the efficacy of prebound antibody toprevent receptor activation. The results of these assays, shown in FIG.4, indicate that phosphorylation of the FLK-1/fms is reduced by thesimultaneous addition of mAb with VEGF and markedly inhibited byantibody prebound to the receptor. A densitometry scan of these datarevealed that mAb DC101 interacts with FLK-1/fms to inhibitphosphorylation to a level that is 6% (lane 5, P) and 40% (lane 6,C ) ofthe filly stimulated receptor control (lane 4). From these data we inferthat mAb DC101 strongly competes with the ligand-receptor interaction toneutralize FLK-1 receptor activation. To determine the sensitivity ofthe VEGF-FLK-1 interaction to inhibition by mAb DC101, C441 cells wereassayed with maximal VEGF levels in the presence of increasingconcentrations of antibody. Assays were performed with the mAb undercompetitive and prebinding conditions. The results of these mAbtitrations are shown in FIG. 9. A marked decrease in the phosphorylationof FLK-1 is observed when mAb DC101 competes with VEGF antibody atconcentrations greater than 0.5 ug/ml. These data also show thatrelatively low concentrations of prebound antibody (<1 ug/ml) aresufficient to completely inhibit receptor activation by ligand.

Example V-6 Activity of DC101 by Phosphorylation Assay

To further evaluate the antagonistic behavior of mAb DC101 on receptoractivation, phosphorylation assays were performed in which a fixedamount of antibody (5 ug/ml) was added to C441 cells stimulated withincreasing amounts of ligand (FIG. 3A). The level of phosphorylationinduced by each ligand concentration in the presence and absence of mAbDC101 was also quantitated by densitometry readings. The plot of thesedata given in FIG. 3B indicates that the antibody was able to partiallyneutralize receptor phosphorylation even in the presence of excessamounts of VEGF. To evaluate the specificity of mAb DC101 on receptoractivation, the antibody was tested for its ability to competitivelyinhibit CSF-1 induced activation of the fms/FLK-2 receptor in the 3T3transfected cell line, 10A2. In these experiments 5 ug/ml of mAb DC101was tested together with CSF-1 concentrations (20-40 ng/ml) that areknown to result in full activation of the receptor. These results, whichare shown in FIG. 10, indicate that mAb DC101 has no effect on the CSF-1mediated phosphorylation of the fms/FLK-2 receptor.

Example V-7 DC101 Inhibition by Pre-Incubation Studies

The extent and specificity of antibody inhibition was further assessedby studies in which DC101 or an irrelevant antibodies were preincubatedwith cells before the addition of ligand to assure maximal interactionof antibody with receptor. In these experiments, monolayers werepreincubated with either 5 ug/ml of DC101, a rat anti-FLK2 mAb (2A13) ora control rat IgG1 (Zymed Labs) prior to the addition of 40 ng/ml ofVEGF. For comparison, competitive assays were run in which antibodiesand VEGF were added simultaneously. The results of these studies showthat only the preincubation of the anti-FLK-1 monoclonal antibody withFLK 1/fins transfected cells completely abrogates receptor activation byVEGF while phosphorylation of FLK1 by VEGF is not affected by theaddition of irrelevant isotype matched rat antibodies. The reactivity ofthe same blot probed with the anti-fms polyclonal (FIG. 11) shows anequal level of receptor protein per lane. These data indicate that thelack of phosphorylation observed with mAb DC101 treated cells was due tothe blockage of a VEGF-induced phosphorylation of equal amounts ofexpressed receptor.

Example V-8 Interaction of Antibodies with Homologous Receptor Forms

Experiments were then conducted to determine whether the anti FLK-1monoclonal antibodies interact with homologous receptor forms on humanendothelial cells. A titration of increasing concentrations of DC101 oncloned HUVEC cells (ATCC) indicated that the antibody displayed acomplex binding behavior. The data represent differential antibodyinteractions with VEGF receptors reported to occur on endothelial cells(Vaisman et al., J. Biol. Chem. 265, 19461 -19466, 1990). Thespecificity of DC101 interaction with VEGF stimulated HUVEC cells wasthen addressed using phosphorylation assays under similar conditions asthose reported for FIG. 8. In these studies DC101 immunoprecipitatesprotein bands from HUVEC cells that have molecular weights similar tothose reported for cross linked VEGF-receptor bands when the ligandcomponent is subtracted (FIG. 12). These bands display an increasedphosphorylation when cells are stimulated by VEGF (compare lanes 1 and 2in FIG. 12). In addition, the VEGF induced phosphorylation of thereceptor bands is potentiated by the inclusion of 1 ug/ml heparin in theassay (lane 3 in FIG. 12). These findings are consistent with previousreports of increased VEGF binding to endothelial cells in the presenceof low concentrations of heparin (Gitay-Goren et al., J. Biol. Chem.267, 6093 -6098.1992)

It is difficult to ascertain which immunoprecipitated protein interactswith DC101 to generate the complex of phosphorylated bands observed inFIG. 12 given the various receptor forms shown to bind VEGF on HUVEC andthe possibility of their association upon stimulation. Cell surfaceexpressed receptor forms with molecular weights of approximately 180(KDR), 155, 130-135, 120-125 and 85 have been reported to bind VEGF onHUVEC. Such findings address the possibility that several differentreceptor forms may heterodimerize upon ligand stimulation in a mannersimilar to that reported for KDR-FLT-1 However, with the exception ofKDR, the exact nature and role of these receptor forms have yet to bedefined. Consequently, antibody reactivity may result frominteraction(s) with one of several VEGF receptors independent of KDR.

DC101 does not react with human KDR in an ELISA format nor bind tofreshly isolated HUVEC by FACS analysis. These results suggest that adirect interaction of DC101 with human KDR is highly unlikely.

Unlike DC101, Mab 25 and Mab 73 both react with human KDR in an ELISAformat and bind to freshly isolated HUVEC by FACS analysis.

Example V-9 Mitogenic Assays of HUVEC

An inhibitory effect of DC101 on endothelial cells was observed when theantibody was tested in mitogenic assays of HUVEC cells (ATCC) stimulatedwith VEGF in the presence and absence of antibody (FIG. 12). Theseresults show that a marked increase in cell proliferation by VEGF isreduced approximately 35% by DC101. Heparin shows no differential effecton cell growth under the growth conditions employed in these assays.

Since DC101 can exert effects on VEGF induced proliferation and receptorphosphorylation of HUVEC it is conceivable that these results are due toa Mab interaction with an undefined receptor form which is poorlyaccessible at the cell surface, but which plays some role, albeit minor,in HUVEC growth. Also, the immunoprecipitation of phosphorylated bandsof the correct molecular weight by DC101 from VEGF stimulated HUVEC alsosupports the notion that DC101 may interact with an undefined FLK-1 likeprotein that associates with an activated receptor complex.

Example V-10 Binding of Mab 25 and Mab 73 to C441 Cells and HUVEC

Mabs 25 and 73 bind to C441 and HUVEC by FACS analysis and showinternalization in both cell lines. Results from western blots show thatboth anti-FLK-1 Mabs can detect the band(s) for the FLK/fms receptor inimmunoprecipitates by an anti-fms polyclonal antibody from C441 cells.(See example IV-2 above for protocol.) These antibodies elicit aspecific neutralization of VEGF induced activation of the FLK-1/fmsreceptor and have no effect on the phosphorylation of the mouse PDGFreceptor by PDGF or the human EGF receptor by EGF. (See example IV-1above for protocol.) They have the capacity to inhibit VEGF stimulatedHUVEC in proliferation assays to 50% whereas DC101 affects growth to afar lesser extent.

Example V-11 Immunoprecipitation of KDR with Mab25 and Mab73

KDR represents one of the phosphoproteins immunoprecipitated by theMab25 and Mab 73 from activated HUVEC. KDR was detected in western blotand immunoprecipitation analyses using an anti-FLK-1/KDR polyclonalantibody (IM142) from VEGF-stimulated early passage HUVEC. Conversely,bands immunoprecipitated by these antibodies from VEGF-stimulated HUVECare cross reactive with IM142 but not an anti-FLT-1 polyclonal antibody.These findings infer that the Mabs may affect the activity of KDR inHUVEC based on experimental evidence implicating KDR as the VEGFreceptor responsible for the proliferative response in activatedendothelial cells (See example IV-3 above for protocol.)

Example VI Presence of VEGF Receptor Forms on Non-Endothelial (Tumor)Cells

Several tumor lines were screened for protein reactivity with DC101 byimmunoprecipitation and detection with antiphosphotyrosine. Immunoblotsfrom the cell lines 8161 (melanoma) and A431 (epidermoid carcinoma)yielded phosphorylated bands with molecular weights of approximately 170and 120 kD. These results indicate that a human VEGF receptor form isexpressed in non-endothelial cells, such as tumor cells.

Similar experiments have shown that a KDR like receptor is expressed inan ovarian carcinoma cell line, OVCAR-3. These cells also appear tosecrete VEGF. Phosphorylated bands are immunoprecipitated by an anti-KDRpolyclonal antibody from VEGF-stimulated OVCAR-3 cells that are reactivewith anti-FLK-1 Mabs by western blotting. Also, bands immunoprecipitatedby the murine Mabs from these cells show cross reactivity with the samepolyclonal antibody. Furthermore certain murine anti-FLK-1 Mabs elicitan inhibitory effect on these cells in proliferation assays. Theseresults demonstrate nonendothelial expression (i.e. on tumor cells) ofhuman VEGF receptor forms. The data from the phosphorylation andproliferation assays also suggest that VEGF can modulate receptoractivity in an autocrine and paracrine manner during tumorigenesis. (SeeExample IV-3 above for protocol.)

Example VII In vivo Studies using DC101 Example VII-1 Inhibition in vivoof Angiogenesis by dc101

In vivo studies were designed to determine if an anti-FLK1 monoclonalantibody would block the growth of VEGF-expressing tumor cells. In theseexperiments, a human glioblastoma multiform cell line was used that hashigh levels of VEGF message and secretes about 5 ng/ml of VEGF growthfactor after a 24 hour conditioning in serum free medium (FIG. 5).

On day zero, athymic nude mice (nu/nu; Charles River Labs) were injectedin the flank with 1-2 million glioblastoma cells. Beginning on the sameday, animals received intraperitoneal injections of either DC 101 andcontrol antibodies (100 ug/animal). The mice received subsequentantibody treatments on days 3, 5, 7, 10, 12, 14, 17, 19, and 21. Animalsreceived injections of 100 ug of either DC101 or a control rat antibodyto the murine FLK2 (2A13) receptor on days 0, 3, 5, 7, 10, 12, 14, 17,19 , and 21 for a total inoculation of 1 mg/animal. Tumors began toappear by day 5 and followed for 50 days. Tumor size was measured dailywith a caliper and tumor volume calculated by the following formula:p/6×larger diameter×(smaller diameter)² (Baselga J. Natl. Cancer Inst.85: 1327-1333). Measurements were taken at least three times per weekand tumor volume calculated as described above. One tumor bearing animalin the DC101 group died early in the study and was not used to determinestatistical significance between the groups.

FIGS. 14A and 14B show a comparison between the DC101 and the control2A13 group of reduction in tumor growth over 38 days in individualanimals. Although all animals developed tumors of varying sizes andnumber during the course of the study, DC101-treated mice showed anoverall delay in tumor progression. One mouse in the DC101 groupremained tumor free until day 49 when a small growth was observed. Eventhen, tumor growth was markedly suppressed. Statistical analysis of thedata was done to assess differences in tumor size between the twogroups. Data was subjected to a standard analysis of covariance wheretumor size was regressed on time with treatment as a covariate. Theresults showed that reduction in tumor size over time for the DC101group was significantly different (p<0.0001) from that seen for 2A13injected mice.

FIG. 15 shows the therapeutic efficacy of DC101 in athymic nude micetransplanted with the human glioblastoma tumor cell line GBM-18, whichsecretes VEGF. Nude mice were injected subcutaneously with GBM-18 cellsand divided into three groups of treatment: a PBS control, an irrelevantrat IgG1 control, and DC101. Treatments were administered simultaneouslywith tumor xenografts and continued for four weeks. The results showedthat GBM-18 tumor growth in DC101 treated nude mice was significantlyreduced relative to controls. This experiment indicates that DC101suppresses tumor growth by blocking VEGF activation of FLK-1 on tumorassociated vascular endothelial cells, and that DC101 has therapeuticvalue as an anti-angiogenic reagent against vascularized tumorssecreting VEGF.

Monoclonal antibodies to FLK-1 receptor tyrosine kinase inhibit tumorinvasion by abrogating angiogenesis. Invasive growth and angiogenesisare essential characteristics of malignant tumors. Both phenomena provedto be suitable to discriminate benign from malignant keratinocytes in asurface transplantation assay. After transplantation of a cell monolayerattached to a collagen gel onto the back muscle of nude mice, all tumorcells initially formed organized squamous epithelia, but only malignantkeratinocytes grew invasively within 2-3 weeks. Both benign andmalignant cells induced angiogenesis. Angiogenic response to malignantcells, however, occurred earlier, is much stronger, and capillary growthdirected toward malignant epithelia. Moreover, in transplants of benigntumor cells, capillaries regressed after 2-3 weeks, whereas malignantkeratinocytes maintain the level of ongoing angiogenesis. The vascularendothelial growth factor (VEGF) and its cognate receptor play a pivotalrole in tumor angiogenesis. The administration ofDC101 disrupted ongoingangiogenesis leading to inhibition of tumor invasion. The antibodyprevented maturation and further expansion of newly formed vascularnetwork, but did not significantly interfere with initial angiogenesisinduction. These results provide evidence that tumor invasion requiresprecedent angiogenesis, and that the VEGF receptors are crucial inmaintaining angiogenesis in this model system.

Example VII-2 Effect of Different Concentrations of DC101 on EstablishedGlioblastoma (gbm-18) Tumors

Athymic mice (nu/nu) were inoculated subcutaneously with GBM-18 (humanglioblastoma multiformae). Antibody therapy was initiated when thetumors reached an average volume of 100-200 mm³. Treatment consisted ofsix injections (twice weekly for 3 weeks) of the following: (i) DC-101at 200, 400 or 800 ug/injection; (ii) an irrelevant isotype matched ratIgG (400 ug/injection); or, (iii) PBS. Tumor volumes were measured witha caliper. Tumor inhibition in the DC-101 groups was found to besignificant (*) vs. the PBS and irrelevant monoclonal antibody groups.

Another experiment demonstrates the effects of the rat anti-FLK-1monoclonal antibody DC101 on the growth of GBM-18 tumors in nude mice.Animals (nu/nu; Charles River Labs; ten animals per group) were injectedsubcutaneously with GBM-18 cells (human glioblastoma [100]; 1 millionper animal) on day 0. Treatments with PBS or DC101 (200 μg perinjection) were begun on day 7 and continued twice weekly for 3 weeks(6×). Graphs show a plot of the mean tumor volumes and regressed datafor each group over time with their respective tumor growth rates(slopes given as λ; solid lines) and 99% confidence limits (dottedlines). The slope of the line for animals treated with DC 101 wassignificantly different from that of PBS (p≦0.01). It is important tonote that an irrelevant rat IgG1 monoclonal antibody (anti-mouse IgA;Pharmigen) had no effect on the growth of GBM-18 xenografts and gaveresults similar to that observed with PBS (data not shown).

Supplemental Enablement

The invention as claimed is enabled in accordance with the abovespecification and readily available references and starting materials.Nevertheless, Applicants have deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852 USA (ATCC) thehybridoma cell lines that produce the monoclonal antibodies listedbelow:

Hybridoma cell line DC101 producing rat anti-mouse FLK-1 monoclonalantibody deposited on Jan. 26, 1994 (ATCC Accession Number HB 11534).Hybridoma cell line M25.18A1 producing mouse anti-mouse FLK-1 monoclonalantibody Mab 25 deposited on Jul. 19, 1996 (ATCC Accession Number HB12152).

Hybridoma cell line M73.24 producing mouse anti-mouse FLK-1 monoclonalantibody Mab 73 deposited on Jul. 19, 1996 (ATCC Accession Number HB12153).

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and the regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromdate of deposit. The organism will be made available by ATCC under theterms of the Budapest Treaty, and subject to an agreement betweenApplicants and ATCC which assures unrestricted availability uponissuance of the pertinent U.S. patent. Availability of the depositedstrains is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

1-28. (Canceled)
 29. A process for preparing a polypeptide thatcomprises an amino acid sequence that specifically binds to anextracellular domain of a flt-1 receptor and neutralizes activation ofthe receptor, the process comprising: culturing cells that express anucleic acid molecule comprising a nucleic acid sequence that encodes anamino acid sequence wherein the amino acid sequence consists of thevariable region of a monoclonal antibody that specifically binds to anextracellular domain of a fit-1 receptor and neutralizes activation ofthe receptor; and isolating the polypeptide from the cultured cells.30-32. (Canceled)
 33. A process for preparing a polypeptide thatcomprises an amino acid sequence that specifically binds to anextracellular domain of a fit-1 receptor and inhibits tumor growth in amammal, the process comprising: culturing cells that express a nucleicacid molecule comprising a nucleic acid sequence that encodes an aminoacid sequence wherein the amino acid sequence consists of the variableregion of a monoclonal antibody that specifically binds to anextracellular domain of a fit-1 receptor and inhibits tumor growth inthe mammal; and isolating the polypeptide from the cultured cells.34-52. (Canceled)